Production of podophyllotoxins using podophyllum

ABSTRACT

A process for the preparation of an adventive embryo of a plant belonging to the genus Podophyllum, comprising the steps of preparing cells of a plant belonging to the genus Podophyllum, incubating the cells in a sugar free medium or a medium containing up to 2% by weight sugar, and then incubating the cells in a medium containing more than 2% by weight of sugar to generate an adventive embryo; a process for the production of a podophyllotoxin compound comprising the steps of preparing an adventive embryo of a plant belonging to the genus Podophyllum, culturing the adventive embryo to generate an adventitious organ, culturing the adventitious organ to produce a podophyllotoxin compound, and recovering the podophyllotoxin compound; and a process for the production of a podophyllotoxin compound, comprising the steps of preparing de-differentiated cells or tissue of a plant belonging to the genus Podophyllum culturing the de-differentiated cells or tissue in a liquid medium to generate a differentiated organ, culturing the differentiated organ on a solid medium to produce a podophyllotoxin compound, and recovering the podophyllotoxin compound.

This application is a divisional of Ser. No. 07/603,361, filed Oct. 26,1990 now U.S. Pat. No. 5,025,343.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to processes for the production ofpodophyllotoxin compounds by culturing Podophyllum, and to thepreparation of an adventive embryo useful for such processes.

2. Description of the Related Art

Perennial plants belonging to the genus Podophyllum are importantfoliage plants, and recently have been found to produce podophyllotoxincompounds and flavonoid compounds which are useful in the fields ofpharmaceuticals and cosmetics, and therefore, have become industriallyvaluable. For example, podophyllotoxin, one of the podophyllotoxincompounds, was found to have an aperitive activity and antimicrobialactivity, and has been used as an aperitive medicament or antimicrobialagent. Moreover, recently, podophyllotoxin has attracted attention as ananti-tumor agent, and the development of anti-tumor agents derived frompodophyllotoxin compounds is underway.

Nevertheless, in the industrial production of the podophyllotoxincompounds many problems arise, as in the industrial production of othersubstances of a higher plant origin. Namely, although to produce suchsubstances it is necessary to obtain a naturally occurring desired plantand to-cultivate the plant, the production of the desired product bycultivating the plant is accompanied by various limitations. Forexample, the productivity of a desired product varies according tovarious conditions, including environmental conditions, and in generalthe productivity is relatively low. Therefore, it is very difficult toconsistently obtain a large amount of Podophyllum plant as an industrialstarting material for the production of podophyllotoxin compounds.

To resolve the above-mentioned problems accompanying the use of theplant, the use of a plant tissue culture has been proposed. In thetissue culture method, cells or tissue such as callus, dedifferentiatedcells, adventive embryo, adventitious organ, or the like are cultured ina medium to accumulate a desired product in the culture medium, whichproduct is then recovered.

General procedures for generating and culturing the above-mentionedplant entities are known, but particular conditions for the generationand culturing of the plant entities differ depending on the plant, andconditions suitable for one particular plant are not necessarilysuitable for another plant. Further, the conditions for the productionof a particular substance are very strict, and conditions suitable for aparticular producer plant, or for a particular product, cannot beautomatically applied to the production of an other product or an otherplant. Therefore, the conditions for the production must beexperimentally determined for each particular plant and each particularproduct.

Japanese Unexamined Patent Publication No. 62-96088 describes a processfor obtaining podophyllotoxin compounds in an amount of 0.0035% byweight relative to the plant mass by culturing an explant of aPodophyllum plant on a solid medium to form a callus, which is thencultured on a solid medium to form a large amount of callus/adventitiousroots, and extracting podophyllotoxin compounds from the roots.According to this method, however, the preparation of a large amount ofadventitious roots is not always easy, and an accumulated amount ofpodophyllotoxin compounds is not always satisfactory.

An adventive embryo is useful as a starting or intermediate material fora plant tissue culture for the production of a desired substance,although it is also industrially useful as a starting or intermediatematerial for the mass-proliferation of plants, in the interests of anefficient breeding and efficient cultivation of the plants, and as amain component of artificial seeds.

Nevertheless, it is very difficult to reproducibly generate an adventiveembryo, and to date, the generation of an adventive embryo has beenreported only for a very limited plant species. Moreover, in most cases,since the generation of an adventive embryo is carried out on a solidmedium, the generation of the adventive embryo takes a relatively longtime and the recovery of the adventive embryo is difficult, resulting inan impracticality for an industrial application.

To obtain an adventive embryo as a starting material of theproliferation and tissue culture of useful plants, various methods havebeen proposed. According to one method, non-differentiated cells of adesired plant are cultured in the presence of plant growth hormones suchas auxin to induce the generation of an adventive embryo. According toanother method, an osmotic pressure of a medium for culturing anon-differentiated cell is changed to induce the generation of anadventive embryo without using plant growth modulators. These methodshowever, can not be universally applied to all plants, since theconditions for the generation of an adventive embryo differ depending onparticular plants. Further, the generation of an adventive embryo ofplants belonging to the genus Podophyllum is not known.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a process for thepreparation of an adventive embryo of a plant belonging to the genusPodophyllum, comprising the steps of:

preparing cells of a plant belonging to the genus Podophyllum;

incubating the cells in a sugar free medium or a medium containing up to2% by weight sugar; and then

incubating the cells in a medium containing more than 2% by weight sugarto generate an adventive embryo.

Moreover, the present invention provides a process for the production ofa podophyllotoxin compound comprising the steps of:

preparing an adventive embryo of a plant belonging to the genusPodophyllum;

culturing the adventive embryo to generate an adventitious organ;

culturing the adventitious organ to produce a podophyllotoxin compound;and

recovering the podophyllotoxin compound.

The present invention still further provides a process for theproduction of a podophyllotoxin compound, comprising the steps of:

preparing de-differentiated cells or a tissue of a plant belonging tothe genus Podophyllum;

culturing the de-differentiated cells or tissue in a liquid medium togenerate a differentiated organ;

culturing the differentiated organ on a solid medium to produce apodophyllotoxin compound; and

recovering the podophyllotoxin compound.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present inventions, any plants belonging to the genusPodophyllum, such as Podophyllum peltatum, P. emodi, P. hexandrum, P.pleianthum, P. versipelle, etc., can be used.

Preparation of cells of Podophyllum plant

The present processes involve a step for preparing Podophyllum plantcells; the cells are usually in the form of a callus or cells in aliquid culture.

The callus can be obtained according to a conventional procedure. Forexample, a leaf, stem, root or the like of Podophyllum plant is washedwith water, sterilized with an ethanol aqueous solution, a sodiumhypochlorite aqueous solution, a benzalconium chloride aqueous solutionor the like, and rinsed with sterile water. Then, if necessary orpreferable, the plant part is cut into sections, and the sterilizedplant parts or sections thus prepared are put on a medium in a flask orpetri dish, which is then incubated. Any medium generally used for aplant tissue culture can be used. Prepared examples of such media areMurashige-Skoog medium, Gamborg B5 medium, Nitsch & Nitsch medium,White's medium and the like, supplemented with vitamins and plant growthmodulators. The plant growth modulators include auxins such asindole-3-acetic acid, α-naphthalene acetic acid,2,4-dichlorophenoxyacetic acid and the like; and cytokinins such askinetin, 6-benzyladenine and the like, and are used alone or incombination. After the plant parts or sections are cultured on theabove-mentioned medium at 20° C. to 30° C. for 1 to 4 weeks, a callus ofPodophyllum plant is formed.

Culturing of the callus on either a solid medium or in a liquid mediumprovides liquid culture cells.

In the present invention, "culturing" includes culturing of Podophyllumplant on a solid medium or in a liquid medium. The solid culturing iscarried out by putting the cells described above on an aqueous gelcontaining the above-mentioned medium components. The aqueous gel ispreferably agar gel, gellan gum gel or the like. The liquid culture is aculture wherein at least a part of cells to be cultured are in contactwith a liquid medium during the culturing, and includes, for example,culturing in an aeration/agitation fermenter, an air-lift fermenter, orin a conical flask on a reciprocating or rotating shaker, and culturingby a paper wick method.

Generation of adventive embryo

According to the present invention, an adventive embryo is generated byculturing cells such as a callus, or liquid culture cells of Podophyllumplant are shortly subjected to a starvation condition to a short enoughextent that the plant cells survive. Generally, to maintain and grow aculture of Podophyllum plant in a living state for a long time, thepresence of a sugar at a concentration of more than 2% by weight ispreferable. To subject the culture to the starvation condition, theculture is incubated in a medium containing up to 2% by weight,preferably up to 1.5% by weight, more preferably up to 1.0% by weight,for example, 0.5% by weight, of sugar. Although a sugar-free medium canbe used in the starvation step, since an incubation for a long time, forexample, for longer than 10 weeks, may result in the death of theculture, an incubation in a sugar-free medium for a long time is notpreferable.

The starvation medium is, for example, Murashige-Skoog medium, GamborgB5 medium, Nitsch & Nitsch medium or the modification thereof optionallysupplemented with additional nutrient source and/or plant growthmodulators. The nutrient source is, for example, casein hydrolyzate andcoconut milk. The plant growth modulators are auxins and cytokinins,which are used alone or in combination, at a concentration of up to 3mM. The incubation is carried out usually for 2 days to 10 weeks,depending on the sugar concentration of the starvation medium and otherfactors.

Following the starvation step, the starved plant culture is cultured ina medium having a normal sugar concentration of, for example, more than2% by weight, more preferably at least 2.5% by weight, for example, 3%by weight.

The above-mentioned medium having a normal sugar concentration is, forexample, Murashige-Skoog medium, Gamborg's B5 medium, Nitsch & Nitschmedium or a modification thereof supplemented with sugar, as well asoptionally with magnesium ions, a nutrient source and/or plant growthmodulators. The magnesium ions accelerate the generation of adventiveembryo. As the nutrient source, casein hydrolyzate and coconut milk maybe used. The plant growth modulators are auxins and cytokinins, whichare used alone or in combination at a concentration of up to 3 mM. Theculturing is usually carried out for 2 days to 5 weeks, to generate anadventive embryo in the culture product.

Throughout the above-mentioned starvation under a sugar deficiency andculturing in a normal sugar concentration, the culturing temperature isabout 10° C. to 30° C.; the sugar is, for example, sucrose, glucose orgalactose; and the culturing is carried out under either light or darkconditions.

According to the present process, an industrially valuable adventiveembryo of Podophyllum plants can be easily produced without thehindrances of a low availability of plants and the natural environment.

The adventive embryo in the culture medium thus obtained can be used asa starting material for the generation of an adventitious organ, or theadventive embryo can be isolated, for example, by tweezers in the caseof a solid culture or by filtration in the case of a liquid culture, andthe isolated adventive embryo can be used to generate an adventitiousorgan, or in other ways.

Generation of adventitious organ

In the present invention, the term "adventitious organ" means tissueshaving a morphology of an adventitious root or adventitious shoot amongthe tissues derived from an adventive embryo by culturing the latter ina liquid medium or on a solid medium.

The adventitious organ can be generated by culturing an adventive embryoof the Podophyllum plant in a suitable medium under suitable cultureconditions. The culturing can be carried out on a solid medium or in aliquid medium. The medium is preferably Murashige-Skoog medium,Linsmaier-Skoog medium, Nitsch & Nitsch medium or Gamboeg B5 mediumsupplemented with vitamins.

The medium preferably contains plant growth modulators, an additionalnutrient source and/or a sugar. The plant growth modulators are auxinssuch as indole-3-acetic acid, e-naphthaleneacetic acid,2,4-dichlorophenoxyacetic acid and the like, and cytokinins such askinetin, 6-benzyladenine and the like. The auxin and cytokinin are usedalone or in combination at a concentration of up to 5 mM. The nutrientsource is casein hydrolyzate or coconut milk. The sugar is, for example,glucose, sucrose or galactose, which is added at a concentration of upto 5% by weight. Alternatively, sugar is not added. The culturing iscarried out at about 10° C. to 30° C. for 2 to 18 weeks, in light ordark conditions.

According to the present invention, as described above, an adventitiousorgan can be generated, by culturing an adventive embryo in a liquidmedium or on a solid medium under conditions suitable for the generationof the adventitious organ. Alternatively, the proliferation of anadventive embryo and generation of an adventitious organ can be carriedout simultaneously.

Growing adventitious organ and production of podophyllotoxin compounds

The adventitious organ thus generated can be grown in a conventionalliquid medium or on a conventional solid medium under a conventionalcondition, for example, under the above-mentioned condition, forexample, for more than two weeks. During the growing, the adventitiousorgan produces and accumulates podophyllotoxin compounds in an amountmore than that existing in the parent plant.

The podophyllotoxin compounds thus produced can be extracted with anorganic solvent for example, an alcohol such as methanol or ethanol, ora ketone such as acetone. The extracted podophyllotoxin can be purifiedaccording to a conventional procedure.

Generation of differentiated organ

In the present invention, the term "differentiated organ" means tissueshaving a morphology of an adventitious root or adventitious shoot amongthe tissues derived from de-differentiated cells or tissue by culturingthe cells or tissues in a liquid medium.

The de-differentiated cells or tissue can be prepared by culturing acallus or tissue in a liquid medium or on a solid medium, as describedabove.

A differentiated organ can be generated by culturing thede-differentiated cells or tissue in a liquid medium, and underconditions suitable for the generation of the differentiated organ. Asuitable medium is Murashige-Skoog medium, Linsmaier-Skoog medium orWhite's medium, preferably supplemented with an additional nutrientsource, a sugar, and/or plant growth modulators. The nutrient source is,for example, casein hydrolyzate or coconut milk. The sugar is added at aconcentration of up to 3% by weight. Alternatively, sugar is not added.The plant growth modulators are auxin and cytokinin at a ratio of 100:1to 1:100, and a concentration of each modulator is preferably up to 1mM. The culturing is carried out at 15° C. to 30° C., for 2 to 10 weeks,in light or dark conditions.

In an embodiment according to the present invention, as described above,a callus or tissue of the Podophyllum plant is prepared; the callus ortissue is cultured in a liquid medium or on a solid medium to obtain alarge amount of de-differentiated cells or tissue; the de-differentiatedcells or tissue are proliferated by culturing same in a liquid medium;and then the proliferated de-differentiated cells or tissue are culturedin a liquid medium suitable for differentiation, to generate adifferentiated organ. Alternatively, in another embodiment, thegeneration and proliferation of de-differentiated cells or tissue, andthe generation of a differentiated organ, can be carried out in one stepby culturing a callus or tissue in a liquid medium.

Growing differentiated organ and production of podophyllotoxin compounds

The differentiated organ thus generated can be grown on a conventionalmedium such as agar medium, paper wick medium or filter paper medium,for example, for more than 2 weeks. During the growing, thedifferentiated organ produces and accumulates podophyllotoxin compoundsin an amount more than that existing in the parent plant.

The podophyllotoxin compounds thus produced can be extracted with anorganic solvent, for example, an alcohol such as methanol or ethanol, ora ketone such as acetone. The extracted podophyllotoxin can be purifiedaccording to a conventional procedure.

According to the present invention, an adventive embryo can be easilyand reproducibly obtained, and the adventive embryo can be proliferatedto a large amount, or can be used to generate an adventitious organ,which is useful for the production of podophyllotoxin compounds.Moreover, a differentiated organ can be generated from de-differentiatedcells or tissue, and the differentiated organ is also useful for theproduction of podophyllotoxin compounds.

EXAMPLES

The present invention will be further illustrated by, but is by no meanslimited to, the following examples.

Example 1 Formation of callus

Rhizome of Podophyllum peltatum was thoroughly washed with water,sterilized with 70% ethanol for two minutes, sterilized with a 1% sodiumhypochlorite aqueous solution for one minute, and rinsed with sterilewater. The sterilized rhizome was cut into sections having a length ofabout 5 mm, in a sterile atmosphere, and the sections were put on aMurashige-Skoog medium containing 3% sucrose, 1% agar, 1mg/lα-naphthaleneacetic acid, 0.2 mg/l kinetin and 500 mg/l caseinhydrolyzate, and cultured by a stationary culture for 5 weeks in thedark, to generate a callus. The callus was passaged several times.

Subjection to low sugar concentration

Next, the thus-prepared callus was put into 100 ml of a liquidMurashige-Skoog medium in a 300 ml flask containing 0.5% by weightsucrose and 500 mg/l casein hydrolyzate, and cultured for 5 weeks at 25°C. in the dark, on a rotary shaker at 130 rpm.

Generation of adventive embryo

Thereafter, the above-mentioned culture was transferred to a liquidMurashige-Skoog medium in a 300 ml flask containing 3% by weightsucrose, 1 mg/l α-naphthaleneacetic acid, 0.02 g/l kinetin, and 500 mg/lcasein hydrolyzate, and cultured for 2 weeks at 25° C. in the dark, on arotary shaker at 130 rpm. In the resulting culture, 30% by weight of thecells were an adventive embryo.

Comparative Example 1

Example 1 was repeated except that, in place of the step of subjectionto a low sugar concentration, the callus was cultured in the same mediumbut containing 3% by weight sucrose. In this case, the callus continuedto grow, and although a large amount of callus was obtained, anadventive embryo was not obtained.

Comparative Example 2

Example 1 was repeated except, that in the step of the generation of anadventive embryo, the same medium but not containing sucrose was used.In this case, the callus died, and an adventive embryo was notgenerated.

Example 2

Rhizome of Podophyllum peltatum was thoroughly washed with water,sterilized with 70% ethanol for two minutes, sterilized with a 1% sodiumhypochloride aqueous solution for one minute, and rinsed with sterilewater. The sterilized rhizome was cut into sections having a length ofabout 5 mm, in a sterile atmosphere, and the sections were put on aMurashige-Skoog medium containing 3% sucrose, 1% agar, 1 mg/lα-naphthaleneacetic acid, 0.2 mg/l kinetin and 500 mg/l caseinhydrolyzate, and cultured by a stationary culture for 5 weeks in thedark, to generate a callus. The callus was passaged several times.

Next, the thus-prepared callus was put into 100 ml of a liquidMurashige-Skoog medium in a 300 ml flask containing 0.5% by weightsucrose and 500 mg/l casein hydrolyzate, and cultured for 5 weeks at 25°C. in the dark, on a rotary shaker at 130 rpm.

Thereafter, the above-mentioned culture was transferred to a liquidMurashige-Skoog medium in a 300 ml flask containing 3% by weightsucrose, 1 mg/l α-naphthaleneacetic acid, 0.02 g/l kinetin and 500 mg/lcasein hydrolyzate, and cultured for 2 weeks at 25° C. in the dark, onrotary shaker at 130 rpm. In the resulting culture, 30% by weight of thecells were an adventive embryo.

The embryo thus-obtained adventive was put on a paper wick medium on aliquid Murashige-Skoog medium containing 3% by weight sucrose, 1 mg/lα-naphthaleneacetic acid, 0.2 mg/l kinetin and 500 mg/l caseinhydrolyzate, and cultured by a stationary culture for 4 weeks in thedark to generate an adventitious organ. The adventitious organ waspassaged to the same medium and cultured for 4 weeks, and during theculturing, the adventitious organ grew to a length of 2 cm.

The adventitious organ was extracted with ethanol, the extract wasassayed by a high performance liquid chromatography (HPLC), and it wasfound that 1.5% by weight per dry matter of podophyllotoxin wasaccumulated.

Example 3

The adventive embryo of Podophyllum peltatum prepared as described inExample 2 was inoculated to 20 ml of a liquid Murashige-Skoog mediumcontaining 3% sucrose, 1 mg/l -naphthaleneacetic acid, 0.2 mg/l kinetinand 500 mg/l casein hydrolyzate in a 100 ml Erlenmeyer flask, andcultured at 25° C. in the dark on a rotary shaker at 30 rpm for 4 weeks,to generate an adventitious embryo. After culturing in the same mediumfor 4 weeks in the light, the adventitious organ grew to a length of 1.5cm.

The adventitious organ was extracted with ethanol, the extract wasassayed by HPLC, and it was found that podophyllotoxin in an amount of0.8% by weight per dry was accumulated.

Example 4

Rhizome of Podophyllum peltatum was thoroughly washed with water,sterilized with 70% ethanol for two minutes, sterilized with a 1% sodiumhypochloride aqueous solution for one minute, and rinsed with sterilewater. The sterilized rhizome was cut into sections having a length ofabout 5 mm, in a sterile atmosphere, and the sections were put on aMurashige-Skoog medium containing 3% sucrose, 1% agar, 1 mg/lα-naphthaleneacetic acid, 0.2 mg/l kinetin and 500 mg/l caseinhydrolyzate, and cultured by stationary culture for 5 weeks in the dark,to generate a callus. The callus was passaged several times.

Thereafter, the above-mentioned culture was transferred to a liquidMurashige-Skoog medium in a 300 ml flask containing 3% by weightsucrose, 1 mg/l α-naphthaleneacetic acid, 0.2 mg/l kinetin and 500 mg/lcasein hydrolyzate, and cultured for 10 weeks at 25° C. in the dark, onrotary shaker at 130 rpm, to generate a differentiated organ. Thedifferentiated organ was cultured on a solid Murashige-Skoog mediumcontaining 1% agar and 500 mg/l casein hydrolyzate, but not includingplant growth modulators, to grow the differentiated organ. The resultingdifferentiated organ was extracted with ethanol, the extract was assayedby HPLC, and it was found that podophyllotoxin was accumulated in anamount of 1.6% by weight per dry matter.

We claim:
 1. A process for the production of a podophyllotoxin compound,comprising the steps of:preparing an adventive embryo of a plantbelonging to the genus Podophyllum by the process comprising the stepsof preparing cells of said plant in the form of a callus or cells in aliquid culture, incubating the cells in a sugar-free medium or a mediumcontaining up to 2%, inclusive, by weight of sugar, and then incubatingthe cells in a medium containing more than 2% by weight of sugar;culturing the adventive embryo to generate an adventitious organ;culturing the adventitious organ to produce a podophyllotoxin compound;and recovering the podophyllotoxin compound by extracting said culturedadventitious organ with an organic solvent.